XMAP215 Phosphorylation and Function In Vivo.

微管（MT）是关键的亚细胞结构，是α-和β-微管蛋白的动态聚合物。 MT在真核细胞的生存中起着重要的结构和运动相关的作用。 卵和胚胎的青蛙，非洲爪蟾，含有几个微管（MT）相关蛋白（MAP）已被假定为调节MT组织和动力学。其中之一，XMAP 215，是进化上古老的MAP 215/Dis 1家族的成员，在真核生物中MT组织和细胞分裂所需的MAP。XMAP 215促进MT组装和动力学在体外和拮抗驱动蛋白家族成员XKCM 1的不稳定作用。最近的研究结果表明，XMAP 215在中心体和纺锤体极的结构和功能中起着重要作用，从而在细胞分裂过程中纺锤体的组织和功能中起着重要作用。 XMAP 215在M期过度磷酸化，先前的结果表明，XMAP 215活性在体外受CDK 1依赖性磷酸化的调节。然而，磷酸化在调节XMAP 215功能，从而MT组装和动力学，在体内的作用还没有得到系统的研究。计算预测表明，XMAP 215可能在多达一百个不同的位点上被磷酸化。在这个项目中，各种生物化学，生物物理学和细胞生物学方法将被用来研究XMAP 215活性的调节在体外和体内的磷酸化，专注于已知的三个激酶的作用，调节MT组织和细胞周期：CDK 1，极光A，和MARK。将解决以下具体问题：1，XMAP 215在体内是否被CDK 1、MARK和/或Aurora A激酶磷酸化？质谱将用于绘制和测序XMAP 215 M的磷酸肽，提供细胞中XMAP 215磷酸化位点的第一份“清单”; 2. XMAP 215中预测的CDK 1靶位点的磷酸化是否在体内调节MT结合和/或功能？定点突变将被用来探测在XMAP 215中发现的两个预测的CDK 1靶标在爪蟾组织培养细胞、卵母细胞和胚胎中的体内功能; 3、极光A的磷酸化是否调节XMAP 215的活性？体外磷酸化、体外功能测定和定点诱变的组合将用于探测Aurora A依赖性磷酸化在体外和体内调节XMAP 215功能中的作用;和4，预测的MARK和/或Aurora A靶点的磷酸化是否调节XMAP 215中心体靶向和/或XMAP 215体内功能？定点诱变将用于探测MARK和Aurora A依赖性磷酸化在XMAP 215靶向爪蟾组织培养细胞、卵母细胞和胚胎中的中心体和MTOC中的作用。建议项目的智力价值：上述实验的结果应该回答一些悬而未决的问题，关于XMAP 215的调节蛋白激酶和磷酸化，并应提供一个地图的网站磷酸化在体内和功能分析的作用，这些网站发挥调节XMAP 215的定位和活动。这些信息将反过来为XMAP 215和微管相关蛋白的进化古老的MAP 215/Dis 1家族的其他成员如何在细胞周期和发育过程中调节MT组装，组织和功能提供新的见解。拟议项目的更广泛影响：拟议项目将通过为至少一名研究生、两名或更多本科生和一名或更多中学教师提供培训和教育（通过犹他州大学的中学教师科学硕士课程），使教育和人力资源开发受益。 间接的教育效益也将通过加德博士对本科生和研究生教学以及大学外展计划的重大贡献来提供。

Microtubules (MTs) are key subcellular structures that are dynamic polymers of alpha- and beta-tubulins. MTs play a number of structural and motility-related roles that are critical for the survival of eukaryotic cells. Eggs and embryos of the frog, Xenopus laevis, contain several microtubule (MT)-associated proteins (MAPs) that have been postulated to modulate MT organization and dynamics. One of these, XMAP215, is a member of the evolutionarily ancient MAP215/Dis1 family of MAPs required for MT organization and cell division in eukaryotes. XMAP215 promotes MT assembly and dynamics in vitro and antagonizes the destabilizing effects of the kinesin-family member XKCM1. Results from recent studies suggest that XMAP215 plays important roles in the structure and function of centrosomes and spindle poles, and thus in the organization and function of spindles during cell division. XMAP215 is hyperphosphorylated during M-phase, and previous results demonstrated that XMAP215 activity is regulated by CDK1-dependent phosphorylation in vitro. However, the role of phosphorylation in regulating XMAP215 function, and thereby MT assembly and dynamics, in vivo has not been systematically studied. Computational predictions suggest that XMAP215 might be phosphorylated on as many as one hundred different sites. In this project, a variety of biochemical, biophysical and cell biological methods will be used to study the regulation of XMAP215 activity in vitro and in vivo by phosphorylation, focusing on the roles of three kinases known to regulate MT organization and the cell cycle: CDK1, Aurora A, and MARK. The following specific questions will be addressed: 1, is XMAP215 phosphorylated by CDK1, MARK, and/or Aurora A kinases in vivo? Mass spectroscopy will be used to map and sequence phosphopeptides of XMAP215M, providing the first "inventory" of the sites phosphorylated on XMAP215 in cells; 2. does phosphorylation of the predicted CDK1 target sites in XMAP215 regulate MT binding and/or function in vivo? Site-directed mutagenesis will be used to probe the in vivo function of the two predicted CDK1 targets found in XMAP215, in Xenopus tissue culture cells, oocytes and embryos; 3, does phosphorylation by Aurora A modulate XMAP215 activity? A combination of in vitro phosphorylation, in vitro functional assays, and site-directed mutagenesis will be used to probe the role of Aurora A-dependent phosphorylation in regulating XMAP215 function in vitro and in vivo; and 4, does phosphorylation of the predicted MARK and/or Aurora A targets regulate XMAP215 centrosomal targeting and/or XMAP215 function in vivo? Site-directed mutagenesis will be used to probe the role of MARK and Aurora A-dependent phosphorylation in the targeting of XMAP215 to centrosomes and MTOCs in Xenopus tissue culture cells, oocytes and embryos. INTELLECTUAL MERIT OF THE PROPOSED PROJECT: Results from the experiments outlined above should answer a number of the outstanding questions regarding the regulation of XMAP215 by protein kinases and phosphorylation, and should provide a map of sites phosphorylated in vivo and functional analysis of the role those sites play in regulating XMAP215 localization and activity. This information will, in turn, provide new insight into how XMAP215, and other members of the evolutionally ancient MAP215/Dis1 family of microtubule-associated proteins, regulate MT assembly, organization, and function during the cell cycle and development. BROADER IMPACT OF THE PROPOSED PROJECT: The proposed project will benefit education and development of human resources by providing training and education for at least one graduate student, two or more undergraduate students, and one or more secondary school teachers (through the University of Utah College of Science's Masters of Science for Secondary School Teachers program). Indirect educational benefits will also be provided through Dr. Gard's substantial contributions to undergraduate and graduate teaching and University outreach programs.